Single molecule detection by laser-induced fluorescence has emerged as a powerful tool for the characterization and measurement of biological processes. Single molecule measurements have been used to investigate enzymatic turnovers, to study the exact step size of molecular motors, and to observe the diffusion and transport of lipids and receptors on cellular membranes. Much has been learned from these single-molecule studies that had been obscured in ensemble measurements of the same processes, including for example, evidence of history dependent conformations of individual enzymes, the precise step size taken by individual motor proteins, and evidence for hindered diffusion created by domain structure within a cellular membrane [1]. In all of these examples, the motion of the molecule under investigation was limited to zero, one, or two dimensions.
While single molecule tracking in two dimensions has contributed fundamentally to an understanding of cell membrane dynamics, organization, and transport [1] it is important that single molecule imaging techniques be extended from 2 dimensions to 3 dimensions because most aspects of life, such as intracellular signaling and trafficking are three dimensional.
An approach for tracking the 3-dimensional motion of a molecule is to use a series of 2-dimensional images that have the optical signature of the molecule and vary in a measurable fashion with its distance perpendicular to the image plane [2, 3, 4, 5]. The extension of single particle tracking in three dimensions using a series of two-dimensional images has been reported. These studies relied on the fact that the optical signal can vary dramatically and measurably with the distance from the image plane of the sample. One such study involved the use of total internal reflection (TIR) excitation. In TIR excitation, an evanescent wave that is created at a TIR interface is used for fluorescence excitation. The excitation intensity falls off exponentially from the TIR interface with a decay constant on the order of about 100 nm. The intensity of an object reports its position in the direction perpendicular to the image plane, while its “XY” position is determined from its position in the image itself. TIR microscopy has been used to determine the three dimensional position of single GFP molecules suspended in a polymer matrix [3]. A problem with the TIR method is that it is limited to observing the motion of objects that are within about 100 nm of an interface that has a mismatch in the index of refraction. For many biological applications, such as tracking intra-cellular transport or diffusion, this limitation is too restrictive.
In an alternative approach to 3-dimensional tracking, two separate image planes are created in a sample space [6]. This effectively doubles the depth of focus of a microscope such that dynamics can be followed over an extended z range. This extension in z can be useful, for example, to follow dynamics above and below a cell membrane interface for trans-membrane events such as exocytosis. A disadvantage of this approach is that reading out an entire CCD chip takes a substantial amount of time. In addition, reading out the entire CCD chip introduces noise as charges are swept from pixel to pixel, and crude image processing must be used to find the molecule of interest. In the time needed to read-out the CCD chip, process the image, and potentially move to follow the z-motion of the molecule, the molecule of interest likely will have diffused entirely out of the focal plane of the microscope.
Enderlein proposed a method of tracking single molecules in two dimensions using a single photon counting avalanche photodiode as the detection source and an excitation laser beam that sweeps a small circle in the image plane of a microscope objective [7, 8]. With each sweep of the excitation laser, the fluorescence intensity would be a maximum at a certain angular value. The microscope stage would then be moved to position the single molecule in the center of the circle of excitation swept by the laser beam. Levi et al. extended Enderlein's proposed method in order to follow 3-dimensional trajectories [9]. The Levi et al. approach involves four circular sweeps of an excitation laser, with two sweeps above and the other two below the particle being tracked. The intensity profile recorded during these sweeps is used to control a piezo-stage to reposition the object closer to the center of the optical probe volume. Levi et al. demonstrated the 3-dimensional tracking of 0.5 μm diameter fluorescent microspheres in glycerol-water mixtures and followed the phagocytosis of protein-coated beads by fibroblasts. While the method seems promising, it appears to be useful for tracking only very bright particles that are decorated with perhaps thousands of fluorescein equivalents. This limitation stems from the duty cycle of the tracking algorithm. Most of the time the excitation laser isn't exciting the molecule; it is searching for it. The limited number of photons detected by a rapid sweep over a single fluorescent molecule makes this method difficult to implement for single molecule applications.
The extension of single molecule tracking 2-dimensional motion to 3-dimensional spatial trajectories will allow the study of intra-cellular spatial and temporal dynamics such as DNA damage repair pathways, cellular signaling, regulatory networks, or protein trafficking. There is presently no apparatus or method for tracking a molecule in three dimensions by the fluorescence output of the molecule.
There remains a need for an apparatus and method for tracking a molecule or particle in three dimensions by the fluorescence output of the molecule or particle.